High Efficiency Drug Repurposing for New Antifungal Agents Jong H. - PowerPoint PPT Presentation

High Efficiency Drug Repurposing for New Antifungal Agents Jong H. Kim 1, *, Kathleen L. Chan 1 , Luisa W. Cheng 1 , Lisa A. Tell 2 , Barbara A. Byrne 3 , Kristin Clothier 3,4 , and Kirkwood M. Land 5 1 Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, USDA-ARS, 800 Buchanan St., Albany, CA 94710, USA; 2 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA; 3 Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine and 4 California Animal Health and Food Safety Laboratory, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA; 5 Department of Biological Sciences, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA. * Corresponding author: jongheon.kim@ars.usda.gov 1 1

Graphical Abstract High Efficiency Drug Repurposing for New Antifungal Agents: Repositioning of marketed/commercial drugs with no known antifungal activities as new antifungal drugs or fungicides Commercial drug library High sensitivity antifungal screening by incorporating chemical probes & mutants Selection of large number of repurposed antifungal drugs 2 2

Abstract: There has been a persistent effort to improve efficacy of conventional antimycotic drugs. However, current antimycotic interventions have often limited efficiency in treating fungal pathogens, especially those resistant to drugs. Considering development of entirely new antimycotic drugs is a capital-intensive and time-consuming process, we investigated an alternative approach termed drug repurposing whereby new utility of various marketed, non-antifungal drugs could be repositioned as novel antimycotic agents. As a proof of concept, we applied chemosensitization as a new screening strategy, where combined application of a second compound, viz., chemosensitizer, with a conventional drug could greatly enhance antifungal efficacy of the drug co-applied. Unlike the conventional combination therapy, a chemosensitizer itself does not necessarily have to possess an antifungal activity, but the chemosensitizer significantly debilitates defense systems of pathogens to drugs, enabling improved identification of antifungal activity of off-patent drugs. Of note, inclusion of fungal mutants, such as antioxidant mutants, could facilitate drug repurposing process by enhancing the sensitivity of antifungal screening. Altogether, our strategy led to the development of high efficiency drug repurposing, which enhances the drug susceptibility of targeted fungal pathogens. Keywords: Antifungal; Chemosensitization; Drug repurposing; Drug resistance; Signaling pathway 3

Introduction Antifungal drug repurposing is the repositioning process of non-antifungal, • marketed drugs (previously approved for treating other diseases) to treat fungal infections, where the modes of action, cellular targets or safety of the drugs are already identified (Stylianou et al. 2014). While drug repurposing has become a viable approach to accelerate new antifungal drug development, this strategy still requires highly sensitive screening systems. The antioxidant system of fungi is a potential target of antifungal agents (Smits • and Brul 2005, Jager and Flohe 2006). Certain natural compounds, such as derivatives of benzoic acid or sulfur-containing compounds, can be redox- active and thus inhibit fungal growth by interfering with cellular redox homeostasis/antioxidant system (Guillen and Evans 1994, Jacob 2006). Antifungal chemosensitization is an intervention strategy, in which co- • application of a certain natural or synthetic compound, viz., chemosensitizer, with a commercial drug augments the efficacy of the drug co-applied (Kim et al., 2012). While a chemosensitizer does not necessarily have antifungal potency, chemosensitization can lead to: (a) the augmentation of antifungal efficacy of commercial drugs co-applied; (b) overcoming fungal resistance to commercial antifungal drugs; and also (c) enhanced inhibition of mycotoxin production by fungi, such as aflatoxigenic Aspergillus parasiticus (Kim et al., 2014). 4

The yeast Saccharomyces cerevisiae is a useful model system for the • identification of antifungal drugs and their molecular targets in view that: (1) the genome of S. cerevisiae has been sequenced and well annotated ( Saccharomyces Genome Database, www.yeastgenome.org), (2) S. cerevisiae gene deletion mutant collections (~6,000 mutants) have proven to be very useful for determining drug mechanism of action (Parsons et al, 2004; Norris et al, 2013; Lee et al, 2014), and (3) many genes in S. cerevisiae are orthologs of genes of fungal pathogens including Aspergillus sp. (Kim et al, 2005). Using the model yeast S. cerevisiae bioassay, we previously identified several • chemosensitizers, which target cellular antioxidant or cell wall integrity systems (See next slide for examples). In this in vitro study, we tried to develop a high-efficiency drug repurposing • strategy for effective control of fungal pathogens. We selected two drugs (aspirin, bithionol) previously investigated, and concentrated on targeting the oxidative stress response system of fungi with redox-active chemosensitizers, viz., 2-isopropyl-5-methylphenol (Thymol), 4-isopropyl-3-methylphenol (Structural analog of thymol) and 3,5-dimethoxybenzaldehyde. The susceptibility of fungi to the candidate drug (Bithionol) could be • enhanced by co-applying with redox-active chemosensitizers. Bithonol also mitigated fludioxonil tolerance of Aspergillus fumigatus antioxidant signaling mutants. 5

Examples of chemosensitizers targeting antioxidant or cell wall systems in fungi (From the model yeast S. cerevisiae bioassay): Compounds) Antioxidant) Cell)wall) References) targets) targets) 2,3$Dihydroxybenzaldehyde3 3 Kim3et3al.3 sod1 Δ ,3 sod2 Δ ,3 glr1 Δ 3 3 (2008)3 trans $Cinnamaldehyde3 3 Kim3et3al.3 sod1 Δ ,3 sod2 Δ 3 (2011)3 2$Hydroxy$4$methoxy$ 3 Kim3et3al.33 slt2 Δ ,3 bck1 Δ 3 benzaldehyde3 (2015)3 2$Hydroxy$5$methoxy$ 3 Kim3et3al.3 sod1 Δ ,3 sod2 Δ 3 benzaldehyde3 (2011)3 4$Methoxybenzoic3acid3 3 Kim3et3al.33 slt2 Δ ,3 bck1 Δ 3 (2015)3 3,5$Dimethoxybenzaldehyde3 Kim3et3al.33 sod1 Δ ,3 sod2 Δ ,3 glr1 Δ 3 slt2 Δ ,3 bck1 Δ 3 (2011,2015)3 2,5$Dimethoxybenzaldehyde3 Kim3et3al.33 sod1 Δ ,3 sod2 Δ 3 slt2 Δ ,3 bck1 Δ 3 (2011,2015)3 Functions of gene products (See also slide #10) : Sod1, Cytosolic superoxide dismutase; Sod2, Mitochondrial superoxide dismutase; Glr1, Glutathione reductase; Slt2, MAPK of cell wall integrity system; Bck1, MAPKKK of cell wall integrity system. 6

Results and discussion Repurposed drug examples : PubMed search in the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) by using the key words “Drug Antifungal Repositioning” (Search date: May 31, 2018) retrieved 70 articles. We re-evaluated the content of the retrieved articles for their relevance to drug screening, and examples are as shown below. Examples)of)repositioned)drugs)possessing)antifungal)activities.) Compounds) Functions) Repositioning) Target)fungi,) References) methods) outcome)) CLSI 1 )M27+A2) Aliskiren) Anti+hypertensive) C.#albicans# Kathwate) drug) protocol) and) Karuppayil) (2013)) Amiodarone)) Antiarrhythmic)drug) High+throughput) C.#neoformans# Butts)et)al.) adenylate)kinase) (2013)) assay.)) EUCAST 2 )protocol) Aspirin) Anti+pain,)fever,)or) C.#neoformans ,) Ogundeji)) ) inflammation)drug) C.#gatti# et)al.)(2016)) Auranofin) Rheumatoid)) CLSI)M27+A3) Candida,# Thangamani) arthritis)drug) protocol) Cryptococcus# ) et)al.)(2017)) Bithionol)) Antiparasitic)drug) High+throughput)) Exserohilum## Sun)et)al.) ATP)content)assays) rostratum ) (2013)) Human) Neurological)) 24+well)plate)assay) A.#fumigatus# Sebastián+) glycogen) disorder)drug) using)five)GSK+3)) Pérez)et)al.) synthase)kinase) inhibitors) (2016)) 3)(GSK+3)) inhibitors) Tosedostat) Anti+cancer) EUCAST)protocol) C.#albicans ,) Stylianou)) (Aminopeptidase) ) C.#glabrata ) et)al.)(2014)) inhibitor))drug)) ) 1 Clinical)&)Laboratory)Standard)Insitute) 2 European)Committee)on)Antimicrobial)Susceptibility)Testing)) ) 7

We chose aspirin and bithionol as representative drugs for further investigation. Aspirin (Acetyl salicylic acid) is a non-steroidal anti-inflammatory agent and • binds to/acetylates serine residues in cyclooxygenases. This drug decreases synthesis of prostaglandin, platelet aggregation, and inflammation (https://pubchem.ncbi.nlm.nih.gov/compound/2244). Bithionol is a halogenated anti-infective agent that is used against trematode • and cestode infestations. This drug inhibits human soluble adenylyl cyclase (Kleinboelting et al. 2016). Octyl gallate (OG) was used as a positive control for antifungal bioassay. The • mechanism of antifungal action of OG was previously determined as: (a) interrupting the lipid bilayer-protein interface in fungal cells, and (b) functioning as a pro-oxidant (redox-active oxidative stressor), thus triggering cytotoxicity in fungi (Kim et al. 2018). 8

Structures of repurposed drugs and OG, (+) Control, tested in this study Aspirin Bithionol Octyl gallate (OG) Fungal signaling system as a target: Meanwhile, oxidative signaling systems, such as mitogen-activated protein kinase (MAPK) signaling pathway, have been served as effective antifungal targets for redox- active drugs or compounds (Kim et al. 2012) (Next page). 9